title: Glycoengineered CD20 antibody obinutuzumab activates neutrophils and mediates phagocytosis through CD16B more efficiently than rituximab.
abstract: Obinutuzumab (GA101) is a glycoengineered type 2 CD20 antibody with enhanced CD16A-binding and natural killer-mediated cytotoxicity. CD16B is highly homologous to CD16A and a major FcγR on human polymorphonuclear neutrophils (PMNs). We show here that glycoengineered obinutuzumab or rituximab bound CD16B with approximately sevenfold higher affinity, compared with nonglycoengineered wild-type parental antibodies. Furthermore, glycoengineered obinutuzumab activated PMNs, either purified or in chronic lymphoblastic leukemia whole blood, more efficiently than wild-type rituximab. Activation resulted in a 50% increase in CD11b expression and 70% down-modulation of CD62L on neutrophils and in release of tumor necrosis factor alpha, IL-6, and IL-8. Activation was not accompanied by generation of reactive oxygen species or antibody-dependent cellular cytotoxicity activity, but led to up to 47% phagocytosis of glycoengineered anti-CD20 opsonized chronic lymphoblastic leukemia targets by purified PMNs. Significant phagocytosis was observed in whole blood, but only in the presence of glycoengineered antibodies, and was followed by up to 50% PMN death. Finally we show, using anti-CD16B and anti-CD32A Fab and F(ab')2 fragments, that both of these receptors are involved in PMN activation, phagocytosis, and cell death induced by glycoengineered antibodies. We conclude that phagocytosis by PMNs is an additional mechanism of action of obinutuzumab mediated through its higher binding affinity for CD16B.
authors.affiliation: Center of Cellular Therapy "G. Lanzani," Division of Haematology, Azienda Ospedaliera Papa Giovanni XXIII, Bergamo, Italy;
authors.forename: Josée
authors.initials: J
authors.lastname: Golay
authors.forename: Fabio
authors.initials: F
authors.lastname: Da Roit
authors.forename: Luca
authors.initials: L
authors.lastname: Bologna
authors.forename: Claudia
authors.initials: C
authors.lastname: Ferrara
authors.forename: Jeanette H
authors.initials: JH
authors.lastname: Leusen
authors.forename: Alessandro
authors.initials: A
authors.lastname: Rambaldi
authors.forename: Christian
authors.initials: C
authors.lastname: Klein
authors.forename: Martino
authors.initials: M
authors.lastname: Introna
mesh_terms: D061067:Antibodies, Monoclonal, Humanized / Q000737:chemistry / Q000276:immunology* / Q000494:pharmacology; D058846:Antibodies, Monoclonal, Murine-Derived / Q000494:pharmacology*; D000915:Antibody Affinity; D018951:Antigens, CD20 / Q000276:immunology; D039481:CD11b Antigen / Q000096:biosynthesis / Q000235:genetics; D005643:Fucose; D058851:GPI-Linked Proteins / Q000276:immunology; D006031:Glycosylation; D006629:Hirudins / Q000494:pharmacology; D006801:Humans; D015850:Interleukin-6 / Q000378:metabolism; D016209:Interleukin-8 / Q000378:metabolism; D019041:L-Selectin / Q000096:biosynthesis / Q000235:genetics; D015451:Leukemia, Lymphocytic, Chronic, B-Cell / Q000097:blood / Q000276:immunology; D018375:Neutrophil Activation / Q000187:drug effects*; D010587:Phagocytosis / Q000187:drug effects*; D015202:Protein Engineering; D020033:Protein Isoforms / Q000276:immunology; D017452:Receptors, IgG / Q000276:immunology*; D011994:Recombinant Proteins / Q000494:pharmacology; D000069283:Rituximab; D020349:Surface Plasmon Resonance; D014409:Tumor Necrosis Factor-alpha / Q000378:metabolism; D015854:Up-Regulation
chemical_list: D061067:Antibodies, Monoclonal, Humanized; D058846:Antibodies, Monoclonal, Murine-Derived; D018951:Antigens, CD20; D039481:CD11b Antigen; C428200:FCGR3B protein, human; D058851:GPI-Linked Proteins; D006629:Hirudins; C508600:IL6 protein, human; C509295:ITGAM protein, human; D015850:Interleukin-6; D016209:Interleukin-8; D020033:Protein Isoforms; D017452:Receptors, IgG; D011994:Recombinant Proteins; D014409:Tumor Necrosis Factor-alpha; D019041:L-Selectin; D005643:Fucose; D000069283:Rituximab; C543332:obinutuzumab; C083544:lepirudin